Neutrophils (PMNs) are powerful anti-microbial cells that are rapidly mobilized and recruited to infected tissues and sites of tissue damage. Once within tissues, PMNs can deploy many activities to remove pathogens and/or damaged cells. Dysregulation of these events can have severe consequences for the host and contribute to the pathology a variety of diseases, including diabetes, thrombus formation and lupus. It has been increasingly recognized that immune cells generate appropriate responses by integrating a variety signals from receptors through multi-protein platforms, called microclusters. This signal integration has been studied extensively in T cells, but the specific rules of integration and cellular outcomes for one cell type do not necessarily apply to other cells because each cell type plays distinct roles in immunity. How PMNs integrate signals from the host and bacteria to generate the correct response in infected tissues is very poorly understood. This application seeks to uncover and understand critical molecular mechanisms PMNs employ to trigger bactericidal responses, using the bacterial pathogen, Yersinia, and its effector proteins, YopH and YopO. Based on our preliminary data indicating that YopH disrupts integration of signals by inactivating SKAP-2, PRAM-1 and SLP- 76-regulated pathways that emanate from micro-clusters, we will focus on understanding the role of SKAP2 in PMN responses. Our central hypothesis is that YopH and YopO impair PMN responses to bacteria by modulating a SKAP2-dependent system that coordinates the anti-microbial functions of PMNs triggered a variety of different types of receptors. Our aims are (1) to determine the critical functions of YopH in tissu infection; (2) dissect the role of SKAP2 in the anti-microbial responses of PMNs; and (3) understand the effects of YopO on neutrophil signaling after receptor stimulation. Through the proposed research, we expect to contribute a better understanding of the molecular mechanisms critical for PMNs anti-microbial responses. An understanding of how PMNs are inactivated by Yops in infected tissues will reveal the mechanisms by which PMNs deploy their anti-microbial and tissue damaging arsenal and lead to new ways to stop this deployment in auto-immune syndromes.